Phosphorylase kinase is an enzyme involved in carbohydrate metabolism in muscle whose activity is regulated by physiological concentrations of Ca++ ions. We have found that Mg++ acts in concert with Ca++ to cause a time-dependent activation of the enzyme. One of the goals of this project is to gain an understanding of the mechanism underlying this synergistic activation. We will determine whether time-dependent conformational changes occur in response to these metals, and if so, whether the rate of activation corresponds to an identifiable conformational state. In order to determine whether there is a salient conformational change associated with activation in general, we will examine the conformational changes produced by other factors that cause activation (phosphorylation and alkaline pH). We will also perform a topological analysis in an attempt to determine the overall conformation of the molecule, the spatial relationships of its sixteen subunits, and whether those spatial relationships are altered by effectors of the enzyme, such as Ca++ plus Mg++. Another Ca++ dependent process which we will continue to study is autoactivation resulting from autophosphorylation. We will further characterize the properties of the autophosphorylated enzyme and will attempt to determine if autophosphorylation occurs in vivo. The sum of all these data should provide information concerning regulation of glycogen breakdown in muscle and how that breakdown, with subsequent energy production, is coupled to muscle contraction.